A high-pressure freezing device of the above-stated type is described in DE 100 25 512 A1, in which additionally a particularly rapid pressure increase is achieved using a prestressed pneumatic cylinder.
A device produced by the applicant for the high-pressure freezing of biological and industrial samples is currently being successfully marketed under the name “Leica EM HPM100”. With the “Leica EM HPM100”, samples can be cooled to low temperatures, such as below −100° C., within only a few ms using liquid nitrogen under a pressure of up to 2100 bar (cryopreservation). In this case, sample have a thickness of up to 200 μm and are vitrified during cryopreservation as a result of the rapid cooling process, i.e., the formation of crystalline ice is largely prevented, since crystallization could deteriorate or even destroy the microstructure of the sample.
In the “Leica EM HPM100”, a sample cartridge is used for holding the sample under high pressure conditions during the freezing process. The sample cartridge is made of high-strength plastic and comprises three components, specifically two semicylinders with a channel, which can be used to conduct cooling fluid, and a support plate having an opening designed to receive the sample. The pressure at the location of the sample is generated by the cooling fluid, which is brought to a pressure of 2100 bar for this purpose.
FIG. 8 shows the principal structure of the high-pressure freezing system 9, which is based upon the “Leica EM HPM100” and can also be used for freezing systems according to the invention. The sample cartridge 89 is introduced into the high-pressure chamber 90 by means of a loading device 95. The high-pressure chamber 90 is sealed for the freezing process by locking the loading device 95 with locking pins 94. The freezing process is implemented by introducing pressurized liquid nitrogen (LN2), which is fed rapidly into the space inside the cartridge 89. For this purpose, the system 9 is equipped with a supply 8 of a cooling medium. In the cooling medium supply 8, LN2 is conducted from a cooling medium tank 81 via a cooling medium pump 82, downstream of which a flow check valve 83 is located, to a pressure intensifier 84 operated with compressed air. Here, the cooling medium LN2 that is used is brought to a target pressure of, e.g., 2100 bar. The outlet of the pressure intensifier 84 is connected to the high-pressure chamber 90 by a supply line via a high-pressure valve 85. The high-pressure valve 85 is designed to rapidly supply pressurized LN2 to the interior of the high-pressure chamber 90 when opened, so that the sample held in the cartridge 89 is high-pressure frozen within a very short time interval. A pressure sensor 91 and a temperature sensor 92 monitor the freezing process. Pressure in the chamber 90 can be released via an outlet opening 93 (outlet with noise damper), to allow the cryopreserved sample to be removed.
The high-pressure chamber also offers the option of supplying an additional liquid (“fill liquid”), e.g., an alcohol (particularly ethanol), wherein said fill liquid is supplied to the high-pressure chamber 90 from a tank 86 via a pump/metering system 87, downstream of which a flow check valve 88 is located. In some applications the alcohol can be used to fill the chamber prior to the freezing process, for example; when the freezing process starts, LN2 displaces the alcohol. In many cases, use of the alcohol offers the advantage of a faster build-up of pressure when the LN2 is fed in. However, use of the fill liquid is not mandatory, especially since various types of samples should not come into contact with alcohol. With the apparatus described here, good cooling results can be achieved even without the use of a fill liquid such as alcohol, in particular.
For specific areas of research and development, it is worthwhile to analyze the behavior of a sample following stimulation with light, particularly from the visible region, particularly by analyzing the state achieved through photostimulation. Analyses of this type are conducted, for example, on biological specimens such as plant cells and cyanobacteria. However, under normal conditions (i.e., under the normal living conditions of the biological parent material, generally at ambient temperature and pressure) the photostimulated state is short-lived, and therefore, additional measures are required to fix the transient stimulated state of the sample. The cryopreservation described in the introductory part is suitable for this purpose; rapidly freezing the sample under high pressure is desirable because it allows biological samples to be vitrified without the formation of ice crystals. With known high-pressure freezing devices, however, irradiation of the sample is possible only outside of the high-pressure chamber, since the interior of the high-pressure chamber is inaccessible or inadequately accessible to light. However, since the process for loading a sample into the high-pressure chamber takes several seconds and is difficult to shorten due to limitations of the equipment, the amount of time required to load a sample is far greater than the lifespan of the light-stimulated state to be analyzed, which is why with known devices it is difficult to impossible to implement cryopreservation fast enough following optical stimulation of the sample.